High sensitivity impedance sensor

ABSTRACT

Disclosed herein are example embodiments of a transformative sensor apparatus that is capable of detecting and quantifying the presence of a substance of interest such as a specified bacteria within a sample via changes in impedance exhibited by a detection electrode array. In an example embodiment, sensitivity is improved by including a focusing electrode array in a rampdown channel to focus a concentration of the substance of interest into a detection region. The focusing electrodes include an opposing pair of electrodes in a rampdown orientation. The focusing electrode may also include tilted thin film finger electrodes extending from the rampdown electrodes. In another example embodiment, trapping electrodes are positioned to trap a concentration of the substance of interest onto the detection electrode array.

CROSS-REFERENCE AND PRIORITY CLAIM TO RELATED APPLICATION

This patent application claims priority to U.S. provisional patentapplication Ser. No. 62/145,842, filed Apr. 10, 2015, the entiredisclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.0022929 awarded by the National Science Foundation and Contract Nos.0020735 and 0040951 awarded by the United States Department ofAgriculture. The government has certain rights in the invention.

INTRODUCTION

Food-borne disease outbreaks can spread quickly and impact largepopulations within a very short period of time. In the United Statesalone, disease outbreaks due to consumption of contaminated food causesan estimated 48 million illnesses, including 128,000 hospitalizationsand 3000 deaths in 2011. These outbreaks have significant impact onhealth and the economy.

The pathogenic strain of Escherichia coli O157:H7 (“E. coli 0157:H7”) isthe most common source for widespread food-borne disease outbreak. E.coli 0157:H7 produces a harmful Shiga toxin that affects intestines,resulting in symptoms that may cause anemia, stomach cramps anddiarrhea. Quick detection of this bacterium in food samples is importantfor containing outbreaks. However, prior detection techniques havesuffered from slowness and low sensitivity.

In an effort to improve upon these shortcomings in the art, theinventors disclose a number of embodiments of a transformative sensorapparatus that is capable of detecting and quantifying the presence ofbacteria of interest within a sample via changes in impedance exhibitedby a detection electrode array.

In an example embodiment, sensitivity is improved by including afocusing electrode array in a rampdown channel upstream from a detectionregion of the sensor apparatus to focus a concentration of the bacteriaof interest into the detection region. The focusing electrodes mayinclude an opposing pair of electrodes in a rampdown orientation. Thefocusing electrode may also include tilted thin film finger electrodesextending from the rampdown electrodes. By applying an alternatingvoltage at a specified frequency to the focusing electrode array, thebacteria of interest can be concentrated toward a desired portion of thefocusing region via dielectrophoresis. The concentrated flow of bacteriacan then be passed into the detection region for detection.

In another example embodiment, trapping electrodes are positioned totrap a concentration of the bacteria of interest onto the detectionelectrode array. By applying an alternating voltage at a specifiedfrequency to the trapping electrodes, the bacteria of interest can beconcentrated onto a detection electrode array via dielectrophoresis.

In still another example embodiment, the focusing electrode array andtrapping electrodes can be combined in the sensor apparatus to furtherimprove sensitivity. For example, an example embodiment of such a sensorapparatus that employs the focusing electrode array in combination withthe trapping electrodes can be capable of detecting the presence of E.coli O157:H7 cells within a sample at concentrations in a range betweenaround 5 CFU/ml to around 10⁴ CFU/mL.

Further still, for other embodiments, substances other than bacteria canbe detected via the inventive sensor technology. For example, thesubstances for detection can be any substance that will contribute to animpedance change by the detection electrodes. By way of example, suchsubstances may include any antigens or substances that elicit antibodyresponses, including but not limited to viruses, viral antigens, fungalantigens, toxins, toxic substances, antibiotic residues. The substancescould also be used to detect antibodies against any antigens orsubstances.

These and other features and advantages of the present invention will beapparent to those having ordinary skill in the art upon review of theteachings in the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an example embodiment of a biosensor apparatus.

FIGS. 2A-2C provide various views of example embodiments of a focusingelectrode array.

FIG. 3A depicts an example embodiment of a detection electrode array incombination with trapping electrode arrays.

FIG. 3B depicts electric field gradients produced by an example set ofdetection electrodes.

FIG. 3C depicts electric field gradients produced by an example set oftrapping electrodes.

FIG. 3D depicts another example embodiment for a trapping electrodearray.

FIGS. 3E-3G depict example dimensions for example embodiments of thesensor apparatus.

FIGS. 3H-3J depict another example embodiment for a trapping electrodearray.

FIG. 4 depicts an example embodiment of a biosensor apparatus thatincludes multiple detection regions in series.

FIG. 5 depicts an example embodiment of a biosensor apparatus thatincludes multiple focusing and detecting regions in parallel.

FIG. 6 depicts another example embodiment of a biosensor apparatus thatincludes multiple focusing and detecting regions in parallel.

FIGS. 7A and 7B depict another example embodiment of a biosensorapparatus that includes multiple detection regions in series.

FIG. 7C depicts another example embodiment of a biosensor apparatus thatincludes multiple detection regions in series.

FIGS. 7D-7F shows simulation views of an example flow through the sensorapparatus of FIG. 7C.

FIGS. 8A-G depict a side view of an example fabrication process for abiosensor apparatus in accordance with an example embodiment.

FIGS. 9A-9H depict an example fabrication process for a biosensorapparatus in accordance with another example embodiment.

FIGS. 10A and 10B depict example embodiments of a three-dimensionalbiosensor apparatus.

FIG. 10C depicts SEM micrographs of the electrodes in the focusing anddetection regions of the apparatus examples shown by FIGS. 10A and 10B.

FIG. 11A depicts an example fabrication process for a biosensorapparatus in accordance with the example embodiments of FIGS. 10A and10B.

FIG. 11B depicts an example electrode fabrication process for abiosensor apparatus in accordance with the example embodiments of FIGS.10A and 10B.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

FIG. 1 depicts an example embodiment of a biosensor apparatus 100 thatis capable of sensing the presence of particles of interest such asbacteria within a fluid material. The apparatus 100 includes a channelthrough which the fluid material flows from an inlet 112 to an outlet116. This channel may take the form of a microchannel. The channelincludes a focusing region 102 and a detection region 104 along a flowpath for the fluid material. The detection region 104 is positioneddownstream from the focusing region 102 with respect to the flowdirection of the flow path.

While the example of FIG. 1 and other examples disclosed below aredescribed as biosensors that detect the presence of bacteria in fluidsamples, it should be understood (as explained above) that substancesother than bacteria or other biological substances can be detected viasuch sensor technology. Accordingly, it should be understood that thesensor technology of FIG. 1 and other figures disclosed herein islabeled as a biosensor by way of example, and other embodiments of thesensor technology disclosed herein can detect non-biological substancesthat would produce an impedance change in the detection electrode array.

Furthermore, in many of the example embodiments discussed below, thebacteria of interest is E. coli 0157:H7. However, it should beunderstood that the inventive technology disclosed herein can also beused to detect other bacteria of interest. For example, the bacteria ofinterest may also be E. coli O26, or E. coli O111. As another example,the inventive technology disclosed herein can be used to detect aplurality of bacteria of the family Enterobacteriaceae. Accordingly, thebacteria of for detection may comprise a bacteria selected from thegroup consisting of Escherichia, Klebsiella, Proteus, Enterobacter,Aerobacter, Serratia, Providencia, Citrobacter, Morganella, Yersinia,Envinia, Shigella, Salmonella, and combinations thereof. As yet anotherexample, the bacteria for detection may comprise a plurality ofBacillus, Campylobacter, Listeria, Staphylococcus, Streptococcus, orVibrio bacteria. Furthermore, the samples being tested can include fluidmaterial corresponding to food products. Examples of such food productsinclude peanut butter, cantaloupes, mangoes, tomatoes, and others.

The focusing region 102 includes a focusing electrode array 106 that isconfigured to create a concentration of the bacteria within the fluidmaterial so that the concentrated mass of bacteria can be directed intothe detection region 104. Through this technique, the concentration ofbacteria within the fluid material exiting the focusing region 102 andentering the detection region 104 will be higher than the concentrationof bacteria within the fluid material entering the focusing region 102from inlet 112. Thus, the focusing region facilitates detection of thebacteria within the fluid material in situations where there may be alow concentration of bacteria present.

In the example of FIG. 1, the focusing electrode array 106 focuses thebacteria concentration toward a center portion of the channel, which hasthe effect of directing the bulk fluid material toward the waste outlets114 that are disposed along an outer portion of the channel. Thedetection region 104 receives the bacteria-concentrated flow of fluidmaterial from the center portion of the focusing region 102.

In the example of FIG. 1, the focusing region 102 is arranged as arampdown channel. As used herein, the term “rampdown” in combinationwith channel refers to an arrangement where the channel is wider at itsinlet than its outlet and progressively narrows along the length of thechannel. For example, the rampdown channel can have dimensions such as alength of around 3 mm, a width at entrance 204 of around 300 μm, a widthat exit 206 of around 100 μm, and a height of around 15 μm. As shown byFIGS. 2A-2C, the focusing electrode array 106 may include a pair ofopposing vertical electrodes 200 that are arranged in a rampdownorientation to define the rampdown channel of the focusing region 102.As shown by FIGS. 2A-2C, the distance 210 between the opposing verticalelectrodes 200 is greater at the entrance 204 to the flow path than thedistance 212 between opposing vertical electrodes 200 at the exit 206from the flow path. The electrodes 200 shown by FIGS. 2A and 2B arecharacterized as “vertical” with respect to a reference system where thehorizontal dimension is the “floor” of the rampdown channel and theelectrodes 200 are the “walls”. However, it should be understood thatthe characterization of which of the dimensions are “horizontal” and“vertical” is arbitrary.

In an example embodiment, the distance 210 between electrodes 200 at therampdown channel inlet 204 can be 80 μm while the distance 212 betweenelectrodes 210 at the rampdown channel outlet 206 can be 10 μm. However,it should be understood that other rampdown widths and ratios could beemployed. Furthermore, an example length of the focusing region 102 canbe a length within the range of 2-4 mm. Once again, it should beunderstood that other lengths could be used.

The electrodes 200 provide focusing effects on the bacteria within thefluid material via dielectrophoresis (DEP). In the example of FIGS.2A-2C, the electrodes 200 are configured to provide positive-DEP (p-DEP)forces via a non-uniform electric field created by applying analternating voltage at a specified frequency to the electrodes 200. Forexample, this voltage signal can exhibit a voltage that falls in a rangebetween around 4-10 Vp-p and a frequency that falls in a range betweenaround 1-10 MHz. This voltage can be applied to the electrodes 200 viathe contacts/bonding pads 120 shown by FIG. 1.

The p-DEP and hydrodynamic forces arising from the rampdown orientationof electrodes 200 operate to push the bacteria toward the center of therampdown channel (e.g., see center line 208) and direct thisconcentration of bacteria in the central portion of the rampdown channeltoward the detection region 104 (see the arrow of center line 208). Thebulk fluid material will keep flowing toward the outer portion of therampdown channel and into waste outlets 114 shown by FIG. 1. Thus, thefluid material entering the detection region 104 will have a higherconcentration of bacteria than the fluid material entering the focusingregion 102 due to the focusing effects created by the electrodes 200 inthe rampdown orientation.

As shown by FIGS. 2A-2C, the focusing electrode array 106 also includesthin film finger pair electrodes 202 extending outward from the verticalwall of each electrode 200. In the example of FIGS. 2A-2C, the fingerelectrodes 202 are orthogonal to the electrodes 200. Thus, in thisexample, finger electrodes 202 are in a horizontal dimension whileelectrodes 200 are in a vertical dimension (such that finger electrodes202 effectively form part of the floor of the rampdown channel whileelectrodes 200 form part of the walls). Moreover, the finger electrodes202 are in a tilted orientation such that opposing finger pairelectrodes are not perpendicular to the center line 208 that defines alongitudinal axis of the rampdown channel. In an example embodiment,these tilted thin film finger pair electrodes 202 are at a 45 degreeangle relative to the center line 208. However, it should be understoodthat other tilt angles could be used.

Thus, for the focusing electrode array 106 shown by FIGS. 2A-2C,initially the tilted thin film finger pair electrodes 202 will generatethe larger p-DEP forces that dominate the focusing process as thealternated voltage is applied to the electrodes 200 and 202 at thespecified frequency. This will focus the bacteria in a narrow line inthe center region of the rampdown channel (e.g., around 8-10 μm wide).As the channel ramps down, the generated p-DEP force from the electrodepairs 200 becomes more dominant relative to the finger electrode pairs202 to dominate the focusing process and push the bacteria of interestin the central portion of the channel toward the detection region 104(see FIG. 2C which shows a simulation of the electric field producedwithin the rampdown channel by the electrodes 200 and 202 in an exampleembodiment).

In an example embodiment, (1) the width of each finger electrode 202 canbe around 10 μm, (2) the spacing between each adjacent finger electrode202 extending outward from the same electrode 200 can be around 10 μm,and (3) the spacing between the distal ends of opposing pairs of fingerelectrodes 202 can be around 8 μm (where the distal ends are the ends ofthe finger electrodes 202 that are opposite the proximal ends thatcontact the electrodes 200). However, it should be understood that othervalues could be employed. Furthermore, an example focusing electrodearray can include 100-200 pairs of finger electrodes 202, although moreor fewer finger electrodes 202 can be employed if desired by apractitioner. It should be understood that the dimensions andorientations of the finger electrodes 202 as well as the dimensions andorientations of the electrodes 200 in concert with the applied voltagecharacteristics can be varied by practitioners to achieve a desiredfocusing effect for a given application.

The detection region 104 includes a detection electrode array 108, withthe detection region being bookended by a trapping electrode array 110.As the concentrated mass of bacteria flows into the detection region,the detection electrode array 108 will exhibit a change in impedance asa function of the bacteria concentration. This impedance change can thenbe measured by an impedance analyzer circuit that is connected to thedetection electrode array 108 via contacts/bonding pads 122 to determineand quantify a presence of bacteria in the fluid material. In an exampleembodiment, the impedance analyzer circuit may take the form of anAgilent impedance analyzer, although it should be understood that othertechniques for measuring impedance could be used. Thus, an impedancetransduction mechanism can be used to detect and quantify bacteria bymeasuring the electrical properties (impedance change) of the bacteriaof interest (e.g., E. coli) caused by binding target molecules (e.g., E.coli) to the receptors (antibodies) immobilized on the surface of thedetection electrode array 108 (discussed below). The magnitude and phaseof the impedance across the detection electrode array 108 can bemeasured as a function of frequency using an impedance analyzer forvarious concentrations in order to determine the lowest measurableconcentration. The testing of each sample concentration will beperformed multiple times (e.g., six times) in order to obtainstatistically viable data. A modulated AC voltage (sine wave) can beapplied to the detection electrode array 108 at a frequency in a rangeof 10 Hz.-10 MHz. The impedance will be measured prior to immobilizingthe antibodies on to the surface of the detection electrode array 108,after immobilizing the antibodies, and after the exposure of bacteria. Apractitioner can use this technique to determine the impedance of thebacteria effect alone. In addition, the effect of frequency on impedancemeasurement can be monitored and analyzed. This technique can establishthe baseline impedance and enable the extraction of the impedance due tothe bacteria of interest alone.

FIG. 3A shows an example embodiment of the detection region 104. Thedetection electrode array 108 may take the form of an interdigitatedelectrode (IDE) array in a horizontal orientation (e.g., along the floorof the channel). The IDE array may comprise opposing pairs of electrodes300 and finger electrodes 302 connected horizontally 300. The IDEelectrode array 300 is disposed longitudinally along the length of thechannel with the fingers 302 connected laterally across the width of thechannel. An example IDE array may comprise 25 pairs of finger electrodes302, although it should be understood that more or fewer fingerelectrodes 302 can be included in the IDE array. Also, in an exampleembodiment, each finger electrode may have a length of around 30 μm anda width in a range of around 5-10 μm, although a practitioner may chooseto employ different lengths and/or widths. The spacing between theopposing interdigitated finger electrodes 302 can be in a range betweenaround 2-10 μm, although once again a practitioner may choose differentspacing amounts. For example, based on modeling results, it is expectedthat miniaturization of the IDE array will significantly increase theimpedance measurement sensitivity, with the spacing between the opposinginterdigitated finger electrodes having greater influence on thestrength of E-field intensity compared with the width of the fingers.FIG. 3B shows a plot of E-field simulation for the IDE array when analternating voltage at a specified frequency is applied across the IDEarray. For example, this voltage signal can exhibit a voltage that fallsin a range between around 4-10 Vp-p and a frequency that falls in arange between around 1-10 MHz.

To promote a binding of the bacteria of interest to the detectionelectrode array 108, a bacteria-specific antibody can be introduced intothe detection region 104. The antibody will coat the surface of thefinger electrodes 302 within the channel, and the bacteria of interestwill bind with the antibody on the finger electrodes 302 to cause theimpedance change that is indicative of the concentration of the bacteriaof interest within the detection region 104. In an example embodimentwhere the bacteria of interest is E. coli 0157:H7, the antibody can bespecific to E. coli 0157:H7. In embodiments where other antigens areused, the antibody can be specific to those antigens in order to promoteattachment. For example, the antibodies can be introduced into thedetection region 104, and once the detection region 104 is filled withthe media, the flow can be stopped for 15-20 minutes, during which timethe antibody will adsorb non-specifically to the surface of thedetection electrode array 108 (e.g., a gold surface of the detectionelectrode array 108 in an example embodiment). Any unbounded antibodiescan be washed using DI water. Next, the corresponding bacteria ofinterest can flow through the detection region, and the bacteria ofinterest will bind to the antibody on the detection electrode array 108due to the specificity of the capture antibody for that bacteria ofinterest.

To further enhance the sensitivity of the biosensor, the trappingelectrode array 110 is employed to more greatly concentrate the bacteriaof interest onto the detection electrode array 108. As shown by FIG. 3A,the trapping electrode array 110 may comprise a first pair of opposingvertical electrodes 310 that are positioned in the detection region 104upstream from the detection electrode array 108 (with reference to theflow direction 314 of fluid material entering the detection region 104of the channel defined by channel walls 304) and a second pair ofopposing vertical electrodes 312 that are positioned in the detectionregion 104 downstream from the detection electrode array 108 (withreference to the flow direction 314).

The trapping electrodes 310 and 312 provide trapping effects on thebacteria within the fluid material via negative DEP (n-DEP). In theexample of FIG. 3A, the electrodes 310 and 312 are configured to producethe n-DEP forces via a non-uniform electric field created by applying analternating voltage at a specified frequency across the trappingelectrode pairs 310 and 312. For example, this voltage signal canexhibit a voltage that falls in a range between around 4-10 Vp-p and afrequency that falls in a range between around 1-10 MHz. This voltagecan be applied to the electrodes 310 and 312 via the contacts/bondingpads 124 shown by FIG. 1. The trapping electrodes 310 and 312 thusgenerate a high E-field gradient that forces the bacteria of interestaway from the trapping electrodes 310 and 312 and toward the portion ofthe channel with a relatively lower E-field gradient. Given thepositioning of the detection electrode array 108 between the trappingelectrodes 310 and 312, this will significantly increase the chance ofplacing the bacteria of interest on top of the detection electrode array108.

The electrodes 310 and 312 can be designed to exhibit an ellipticalshape as shown by the example of FIGS. 3A and 3C. In an exampleembodiment, the elliptical electrodes 310 and 312 can also exhibit alength of 100 μm and a height of 10 μm, although it should be understoodthat different lengths and/or widths could be used. As shown by thesimulation plot of FIG. 3C, the non-uniform electric field gradient ishighest in the channel region that is between the closest parts of theopposing electrode pairs, and the gradient decreases as the distancebetween opposing electrode pairs increases and as one moves along thelength of the channel away from the opposing electrode pairs. Thevoltage applied at a specific frequency to the trapping electrodes 310and 312 polarizes the bacteria of interest (e.g., E. coli cells (or anydielectric particles)) such that they will exhibit n-DEP behavior. Thus,they will be forced to move away from the vicinity of the trappingelectrodes 310 and 312 toward the detection electrode array 108 and betrapped there. It is noted that the use of vertical electrodes for thetrapping electrodes 310 and 312 also results in a non-uniform E-fieldgradient across the height of the electrodes 310 and 312 and thusfurther improving cell trapping mechanism. Furthermore, it should beunderstood that a practitioner can further adjust or optimize thetrapping characteristics of electrodes 310 and 312 through changes tothe shape and/or dimensions of the electrodes 310 and 312 as well aschanges to the voltage and frequency of the signal applied to electrodes310 and 312. For example, any shape that will cause the electrodes 310and 312 to generate a non-uniform electric field may be used (see, forexample, FIG. 3D which shows an example embodiment where the trappingelectrodes 320 exhibit a triangular shape such that the apex of eachopposing triangular electrode 320 is closest to the microchannel whilethe base of each opposing triangular electrode 320 is farthest from themicrochannel).

Returning to the example embodiment of FIG. 1, the focusing region 102can have a width at its entrance of around 300 μm and a width at itsexit of around 100 μm. The focusing region 102 may also have a length ofaround 3 mm and a height of around 20 μm or a value in a range betweenaround 15-30 μm. In the example embodiment, the three branches thatsplit off from the exit of the focusing region 102 can be dimensionedsuch that the center channel which serves as the detection region 104can have a width of around 33 μm, while the two outer channels thatserve as waste outlet branches can each have a width of around 34 μm.The length of the detection electrode array 108 within detection region104 can be around 400 μm, and the length of each trapping electrode canbe around 124 μm. Examples of this are shown by FIGS. 3E-3G However, itshould be understood that other values for these dimensions could beemployed by a practitioner.

Furthermore, in another example embodiment, the trapping electrodes canbe positioned to sandwich the detection electrode array 108 rather thanbookend the detection electrode array. FIGS. 3H-3J show examples of suchan arrangement. In these examples, the trapping region comprises anopposing pair of elliptically shaped vertical electrodes 350 that arepositioned laterally outside the detection electrode array 108 (wherethe longitudinal direction corresponds to the flow direction through themicrochannel) to effectively sandwich the detection electrode array 108,as shown by FIGS. 3H and 3I (where FIG. 3I shows example dimensions thatcan be used). Furthermore, FIG. 3J shows a zoomed-in view of the exampleembodiment of FIG. 3I. These electrodes 350 generate a high electricfield that forces the substance of interest to move toward the region ofhigh E-field using p-DEP in order to ensure an accumulation of thesubstance of interest on the detection electrode array 108.

FIG. 4 depicts an example embodiment of a biosensor apparatus where thedetection region 104 includes multiple detection regions 400, 402, and404 in series. Each series detection region i includes its own detectionelectrode array 108 _(i) (for example, detection region 400 includesdetection electrode array 108 ₁, detection region 402 includes detectionelectrode array 108 ₂, and detection region 404 includes detectionelectrode array 108 ₃). Each detection electrode array 108 i hasassociated contacts/bonding pads 122 i through which it connects with animpedance analyzer circuit. Each of the detection regions 400, 402, and404 is separated from the adjacent detection region via trappingelectrodes. Thus, detection region 400 is positioned between trappingelectrodes 310 and 312, detection region 402 is positioned betweentrapping electrodes 312 and 410, and detection region 404 is positionedbetween trapping electrodes 410 and 412. Also shown by FIG. 4 is anantibody inlet 420 through which antibodies can be introduced into thedetection regions 400, 402, and 404 in order to promote attachment ofthe bacteria of interest to the detection electrode arrays 108 ₁, and108 ₂, and 108 ₃.

The biosensor apparatus of FIG. 4 can be described with reference to anexample use case where the apparatus is used to focus, trap, and detectlow concentrations of E-coli within a fluid material with a volume onthe order of pico liters. The sensor apparatus of FIG. 4 can be used toachieve a rapid detection with high selectivity for accurateidentification of E. coli O157:H7 and sensitivity at a lowconcentration, 10 CFU/ml. The E. coli cells will be introduced via thefluidic inlet 112 into the focusing region that includes the focusingelectrode array 106 (which uses a ramp down vertical electrode pairalong with tilted thin film finger pairs (45°) with a ramp down channel)that generates p-DEP forces to focus and concentrate the cells into thecenter of the microchannel, and direct it toward the detection regionmicrochannel which has a diameter as small as one-third of the focusingregion channel with a nano liter volume. As the fluid exits the focusingregion, the bulk fluid will keep flowing toward the outer channel intothe waste outlets 114 while the concentrated mass of cells flows intothe detection region. The solution with the concentration of E. colisubsequently enters into a narrower detection region channel whichcontains a series of 3 trapping and detection electrodes as discussedabove. Once the detection region channel is full with the solution, thetrapping electrodes 310, 312, 410, and 412 can be turned on for 15-20minutes in order to trap the E. coli on top of the detection electrodes108 ₁, and 108 ₂, and 108 ₃. This region is connected to the outlet 116.As discussed above, the voltage at a specific frequency applied to thetrapping electrodes will generate n-DEP forces that push the E. colicells to the region of low E-field gradient and trap them on top of thedetection electrodes 108 ₁, and 108 ₂, and 108 ₃. This trappingmechanism will facilitate the binding process between E. coli (antigens)to E. coli (antibody, introduced via inlet 420) selectively on the IDEarray(s). After turning off the trapping electrodes, the unboundantigens and other unwanted particles will be washed away using DIwater. The impedance as a function of frequency will be recorded fromeach IDE array separately by an impedance analyzer. Through thisimpedance measurement, one can detect E. coli in the solution with aconcentration of 10 CFU/ml with a high sensitivity and selectivitywithin 1 hour. The use of the series of 3 trapping and detection regionsalong the microchannel such that each electrode array records theimpedance of E-Coli separately is expected to improve the sensor'ssensitivity. The use of these arrays help ensure the capturing of E-Colicells by one or more detection electrode arrays. When E. coli binds toantibodies, only a region of 2-4 μm above the sensor surface will bemodified. The biosensors will then be washed in DI water to remove theunbound or weakly bound E. coli cells to the immobilized antibodies andcleans the debris (such as salts) from the sensor interior surface.

The biosensor apparatus of FIG. 4 can be fabricated using techniquesshown in connection with FIGS. 8A-G. In this example, the biosensorapparatus can be fabricated on a glass substrate 800 using a series ofphotolithography, electroplating, and surface micromachining in thefollowing steps:

-   -   1) Thin films of Chromium (Cr) (804) and Goal (Au) (806) are        sputtered on a glass substrate (800) coated with a thin layer of        SU-8 2005 (802) (see FIG. 8A).    -   2) The Au (806) layer is patterned on the SU-8 2005 (802) layer        to create the electrodes interdigitated electrode arrays,        traces, bonding pads, and seed layer for electroplating the        focusing electrode (see FIG. 8B).    -   3) A photoresist mold (e.g., AZ 4620 (810) is patterned for Au        (see FIG. 8C).    -   4) Au (806) is then electroplated to create the vertical side        wall electrodes (see FIG. 8D).    -   5) The photoresist mold (810) is removed and the Cr (804) layer        is etched 9 (see FIG. 8E).    -   6) SU-8 2025 (808) is spincoated and patterned to create the        microfluidic channel. Also, Au (806) is deposited to create the        trapping electrodes (see FIG. 8F)    -   7) A PDMS (812) cover with inlet/outlet holes for the fluidic        connectors 814 is cured and bonded to the microchannel layer        using an O₂ plasma technique (see FIG. 8G).

FIG. 5 depicts an example embodiment of a biosensor apparatus thatincludes multiple focusing regions 102 and multiple detection regions104 in parallel. A flow of the fluid material is split into multiplepaths upstream from the focusing regions, and each path includes afocusing region 102 followed by a downstream detection region, as shownby FIG. 5.

FIG. 6 depicts another example embodiment of a biosensor apparatus thatincludes multiple focusing regions 102 and multiple detection regions104 in parallel. In the example of FIG. 6, the focusing region 102includes a plurality of focusing electrode arrays 106 in series (e.g., 2focusing electrode arrays 106 in series). In this example pathogendetection system, the sensitivity is increased by increasing the ratioof pathogen bacteria cells to test media volume. The ratio is increaseddrastically by getting rid of 80% volume of the test media. This isachieved by sequential focusing of bacteria cells towards the center ofthe microchannel with the help of two stages of the focusing electrodearrays 106 within each path. Each focusing electrode array 106 can beconfigured as discussed above in connection with FIGS. 2A and 2B. Usingthe DEP principle and the unique configuration of thick-thin electrodes(e.g., where the thickness/height of the microchannel is 15 μm), eachfocusing electrode array 106 generates a non-uniform distribution ofelectric field across the channel width. This along with fluidic dragforce, helps focus the pathogen bacteria cell towards the center of themicrochannel.

The initial width of the channel at the inlet/port can be 500 μm in theexample of FIG. 6. The inlet channel is then divided into five equalpaths, each with 100 μm width. After the 1^(st) focusing electrode array106, each path is further split into three channels with the outer twochannels and the center channel being 30 μm and 40 μm in width,respectively. The outer two channels are connected to the main wasteoutlet-port to get rid of extra test media. The center channel carriesthe focused bacteria cells through the 2^(nd) focusing electrode array106. After the 2^(nd) focusing electrode array, the 40 μm center channelis once again divided into three channels to get rid of more test media,while concentrating the bacteria towards the center of the microchannel.The outer two channels and the center channel being 12 μm and 16 μm inwidth, respectively. The 12 μm wide outer channels are also connected tothe main waste outlet-port. The 16 μm center channel carries theconcentrated bacteria cells towards the detection electrode array 108for that path.

The detection electrode arrays 108 are functionalized with pathogenbacteria specific antibodies and configured as discussed above inconnection with FIG. 3A. As the bacteria come in contact with theantibody, they bind to the surface of the interdigitated electrodedecreasing the exposed surface of the electrodes. Decreased surface areaof the detection electrodes reduces the overall capacitance of thesystem and results in an increase of impedance. The concentration ofpathogen bacteria cell present in the test media can be correlated tothe change in impedance.

FIGS. 7A and 7B depict another example embodiment of a biosensorapparatus 700 that includes multiple detection regions in series and iscapable of detecting different kinds of bacteria. In this example, eachof the four different detection electrode arrays 108 ₁, 108 ₂, 108 ₃,and 108 ₄ can be coated with four different kinds of antibodiesintroduced via antibody inlets 706, 708, 710, and 712 respectively. Thenthe sample comes into the detection channel through the focusingelectrode array 106 after unwanted waste has been filtered out (intowaste outlet 704) and a concentration of the bacteria is passed into thedetection region. By appropriately applying pressure, one can preventcross-contamination of the four different antibodies. For example, twodifferent antibodies can be delivered via ports 706 and 712. Negativepressure is then applied from ports 702 and 718 which causes (1) theantibody introduced via port 706 to come into contact with detectionelectrode array 108 ₁ while the negative pressure prevents that antibodyfrom coming into contact with detection electrode array 108 ₂, and (2)the antibody introduced via port 712 to come into contact with detectionelectrode array 108 ₄ while the negative pressure prevents that antibodyfrom coming into contact with detection electrode array 108 ₃. Then, twomore different antibodies can be delivered via ports 708 and 710 whilenegative pressure is applied from port 716. The negative pressureapplied from port 716 causes (1) the antibody introduced via port 708 tocome into contact with detection electrode array 108 ₂ while thenegative pressure prevents that antibody from coming into contact withdetection electrode arrays 108 ₁ and 108 ₃, and (2) the antibodyintroduced via port 710 to come into contact with detection electrodearray 108 ₃ while the negative pressure prevents that antibody fromcoming into contact with detection electrode array 108 ₂ and 108 ₄.

After the detection electrode arrays 108 have been properly coated withtheir respective antibodies, the antigen sample can be introduced viainlet port 702. Negative pressure is applied at outlet port 718 whilekeeping the other outlets/inlets closed to cause the antigen sample topass through the focusing electrode array 106 and the four detectionelectrode arrays 108. Thus, it can be seen that a design such as thatshown by FIGS. 7A and 7B can be used to test the same sample fordifferent bacteria of interest via the same sensor apparatus 700.

FIG. 7C depicts another example embodiment of a biosensor apparatus 750that includes multiple detection regions in series and is capable ofdetecting different kinds of bacteria. Unlike the example of FIGS. 7Aand 7B, the different detection regions 108 are not aligned on the samelongitudinal axis of a straight line flow path. In the example of FIG.7C, detection region 108 ₁ is aligned with the focusing region, but anangled branch 752 splits off from the exit of detection region 108 ₁,which then again angles into detection region 108 ₂. It can also be seenthat port 752 can serve as both an antibody inlet and a waste outletwith respect to detection electrode region 108 ₁. An antibody inletfeeds into and is aligned with detection region 108 ₂ while a wasteoutlet meets the exit of detection region 108 ₂ at an angle. Alsomeeting the exit of detection region 108 ₂ at an angle is the entranceto detection region 108 ₃. An antibody inlet feeds into and is alignedwith detection region 108 ₃ while a waste outlet is aligned with theexit from detection region 108 ₃.

The sensor apparatus 750 of FIG. 7C works first by injecting a specificantibody from a corresponding inlet to be immobilized on the detectionregions, where the measurement is conducted. This unique series designallows the use of multiple antibody types on the same channel byincorporating three different inlets and three different outlets withthe main channel with specific arrangement to prevent the mixture of theantibody types. As with the example of FIGS. 7A and 7B, selectivepressurization can be used to prevent cross-contamination of antibodiesinto the different detection electrode regions 108 i. Once the antibodytypes settle down, the antigen sample will be injected and the focusedto the detection channel and get rid of the bulk flow. To collect theantigens after testing is complete, water can be injected from the wasteports for detection electrodes 108 ₁₋₃. The antigens can be collectedvia antibody inlet for detection electrodes 108 ₂ and 108 ₃ and sampleinlet for detection electrode 108 ₁. After the antigen bonded with theantibody, the impedance of the electrodes will be measured and thedifference in impedance presents the existence of the antigen sample.

FIGS. 7D-F shows simulation views of an example flow through the sensorapparatus 750. FIG. 7D shows the flow of the sample via flow path 760.For this example flow, all inlets were closed with the exception of thelast outlet. FIG. 7E shows the flow path for the introduction of anantibody, and there areas of no flow are shown by 762. As can be seen,these areas 762 present cross-contamination of the different detectionelectrode regions. FIG. 7F shows a zoomed view of FIG. 7E.

In another example embodiment, the biosensor apparatus may comprise afocusing region with the rampdown channel and rampdown electrodesfollowed by a detection region that includes a plurality of detectionelectrode arrays in series. In this example embodiment, the trappingelectrode arrays may be omitted. FIGS. 9A-9H depict an examplefabrication process for such a biosensor apparatus. FIG. 9A shows a stepof physical vapor deposition of Cr/Au on a SU-8 2005 layer to create asputtered thin film Au surface. FIG. 9B shows a next step of wet etchingof Au to pattern the electrode structures, including the focusingelectrode array and the detection electrode arrays. FIG. 9C shows a stepof spincoating and patterning AZ 4620 to create a mold for Auelectroplating. FIG. 9D shows a step of electroplating Au to create avertical side wall electrode for the focusing electrode array. FIG. 9Eshows a step of removing the AZ 4620 electroplating mold. FIG. 9F showsa step of wet etching of Cr to complete the patterning of the electrodestructure. FIG. 9G shows a step of spincoating SU-2025 to form andpattern the microchannel layer. FIG. 9H shows a step of bonding a PDMScover and fluidic connectors to the substrate to yield the biosensorapparatus.

While the example embodiments described above can be characterized astwo-dimensional (2D) biosensors due to the electrodes being orientedalong two dimensions (horizontal and vertical), it should be understoodthat embodiments of the inventive biosensor designs can also be 3D. Forexample, FIGS. 10A and 10B depict example embodiments of athree-dimensional biosensor apparatus.

FIG. 10A shows a biosensor apparatus 1000 that is designed to detect thepresence of Salmonella in a sample. The focusing region/zone includes aninterdigitated electrode array 1002 that fully surrounds themicrochannel as shown by FIG. 10A. In this example embodiment, thefocusing interdigitated electrode array 1002 employs n-DEP toconcentrate the Salmonella toward the central region of the channelwhile diverting the bulk fluid to the outer region of the channel.Control over whether the electrodes generate n-DEP or p-DEP can beachieved by changing the frequency of the voltage signal applied to theelectrodes. The permeability of the substance of interest and the mediain which the substance resides as well as the frequency of operationaffect the decision as to whether n-DEP or p-DEP should be used todirect the substance of interest in a desired manner. As n-DEP forcesthe Salmonella toward the central region of the channel where theelectric field will be low, the concentrated flow of Salmonella proceedsalong the center channel into the detection region while the bulk fluidis diverted into the outer waste outlets. The detection region/zone alsoincludes an interdigitated electrode array 1004 that fully surrounds themicrochannel as shown by FIG. 10B. A Salmonella-specific antibody can beintroduced into the detection region via fluidic inlets in order topromote attachment of the Salmonella to the detection electrodes.

FIG. 10B shows another biosensor apparatus 1010 that is designed todetect the presence of Salmonella in a sample. The focusing region/zoneincludes an interdigitated electrode array 1012 that fully surrounds themicrochannel as shown by FIG. 10B. In this example embodiment, thefocusing interdigitated electrode array 1012 employs p-DEP toconcentrate the Salmonella toward the outer region of the channel whilediverting the bulk fluid to the center region of the channel. As notedabove, the p-DEP can be created by changing the frequency of the voltageapplied to the electrodes. The concentrated flow of Salmonella isdiverted into the path branches from the outer region of the channelinto the detection region while the bulk fluid is diverted into acentral waste outlet. The detection region/zone also includes aninterdigitated electrode array 1004 that fully surrounds themicrochannel as shown by FIG. 10B. A Salmonella-specific antibody can beintroduced into the detection region via fluidic inlets in order topromote attachment of the Salmonella to the detection electrodes.

FIG. 10C depicts SEM micrograph views of the electrodes in the focusingand detection regions of the apparatus examples shown by FIGS. 10A and10B, where the view of FIG. 10B is a zoomed in perspective view of theview shown by FIG. 10A.

FIG. 11A depicts an example fabrication process for a biosensorapparatus in accordance with the example embodiments of FIGS. 10A and10B, showing the progression of device fabrication from steps (a)through (h). FIG. 11B depicts an example electrode fabrication processfor a biosensor apparatus in accordance with the example embodiments ofFIGS. 10A and 10B. Frame (a) of FIG. 11B shows a patterned bottominterdigitated electrode array, and frame (b) of FIG. 11B shows apatterned photoresist sacrificial microchannel across the interdigitatedelectrode fingers. The middle two frames of FIG. 11B show an opticalimage and SEM micrograph of the photoresist sacrificial channel on theinterdigitated electrode array. The bottom two frames of FIG. 11B showan SEM micrograph of the patterned interdigitated electrode fingers ontop of the sacrificial microchannel.

While the present invention has been described above in relation toexample embodiments, various modifications may be made thereto thatstill fall within the invention's scope, as would be recognized by thoseof ordinary skill in the art. Such modifications to the invention willbe recognizable upon review of the teachings herein. As such, the fullscope of the present invention is to be defined solely by the appendedclaims and their legal equivalents.

What is claimed is:
 1. An impedance-based sensor apparatus comprising: a channel adapted to provide a path for a flow of fluid material that comprises a substance, wherein the channel includes a first channel region and a second channel region, wherein the second channel region is downstream from the first channel region with respect to the flow path for the fluid material, and wherein first channel region is arranged as a rampdown channel; a first plurality of electrodes positioned in the first channel region, the first plurality of electrodes configured to generate a non-uniform electric field within the first channel region that produces a dielectrophoresis effect on the substance to thereby focus a flow of the fluid material into the second channel region that has a higher concentration of the substance relative to the fluid material at an inlet to the first channel region; a second plurality of electrodes positioned in the second channel region, the second plurality of electrodes configured to exhibit a change in impedance based on a concentration of the substance in the fluid material within the second channel region; and a third plurality of electrodes, the third plurality of electrodes configured to generate a non-uniform electric field within second channel region that produces a dielectrophoresis effect on the substance within the second channel region to trap a concentration of the substance in the second channel region for detection by the second plurality of electrodes; wherein the third plurality of electrodes comprise a first pair of opposing electrodes upstream from the second plurality of electrodes with respect to the flow path for the fluid material and a second pair of opposing electrodes downstream from the second plurality of electrodes with respect to the flow path for the fluid material; and wherein the first and second pairs of opposing electrodes exhibit an elliptical shape.
 2. The apparatus of claim 1 wherein the first plurality of electrodes comprise an opposing pair of electrodes in a rampdown orientation that define the rampdown channel.
 3. The apparatus of claim 2 wherein the first plurality of electrodes further comprise a plurality of opposing finger pair electrodes that extend outward from the opposing pair of electrodes in the rampdown orientation.
 4. The apparatus of claim 3 wherein the opposing finger pair electrodes are orthogonal to the opposing pair of electrodes in the rampdown orientation.
 5. The apparatus of claim 3 wherein the opposing finger pair electrodes are tilted at an angle such that the opposing finger pair electrodes are not perpendicular to a center line of a longitudinal axis for the first channel region.
 6. The apparatus of claim 5 wherein the opposing finger pair electrodes include distal ends away from the opposing pair of electrodes in the rampdown orientation, and wherein the opposing finger pair electrodes are positioned in the first channel region such that there is a gap in the first channel region between the distal ends of the opposing finger pair electrodes.
 7. The apparatus of claim 3 wherein the first plurality of electrodes are configured to receive an alternating voltage at a specified frequency to generate the non-uniform electric field within the first channel region and thereby produce a positive dielectrophoresis effect on the substance in the first channel region that causes a higher concentration of the substance within a central region of the first channel region than an outer region of the first channel region, wherein the second channel region is positioned to receive a flow of the fluid material from the central region of the first channel region, and wherein the first channel region includes a waste outlet in the outer region that diverts the fluid material in the outer region away from the second channel region.
 8. The apparatus of claim 1 wherein the third plurality of electrodes are configured to receive an alternating voltage at a specified frequency to generate the non-uniform electric field within the second channel region and thereby produce a negative dielectrophoresis effect on the substance within the second channel region that causes a higher concentration of the substance to be positioned near the second plurality of electrodes.
 9. The apparatus of claim 1 wherein the second plurality of electrodes comprise an interdigitated electrode array.
 10. The apparatus of claim 1 wherein the substance comprises an antigen, and wherein the second channel region includes an inlet for introducing an antibody into the second channel region.
 11. The apparatus of claim 1 wherein the third plurality of electrodes comprise a pair of opposing electrodes that sandwich the second plurality of electrodes in the second channel region such that (1) a first of the second plurality of electrodes is positioned between a first of the third plurality of electrodes and the flow path through the second channel region, and (2) a second of the second plurality of electrodes is positioned between a second of the third plurality of electrodes and the flow path through the second channel region.
 12. The apparatus of claim 11 wherein the first plurality of electrodes comprise an opposing pair of electrodes in a rampdown orientation that define the rampdown channel.
 13. The apparatus of claim 12 wherein the first plurality of electrodes further comprise a plurality of opposing finger pair electrodes that extend outward from the opposing pair of electrodes in the rampdown orientation.
 14. The apparatus of claim 1 wherein the second channel region comprises a plurality of detection regions, each detection region including its own subset of the second plurality of electrodes.
 15. The apparatus of claim 14 wherein a plurality of the detection regions are arranged in series.
 16. The apparatus of claim 14 wherein a plurality of the detection regions are arranged in parallel.
 17. The apparatus of claim 14 wherein the detection regions are adapted for selective pressurization of the detection regions.
 18. The apparatus of claim 14 wherein the third plurality of electrodes comprise a plurality of pairs of opposing electrodes that separate the detection regions.
 19. The apparatus of claim 14 wherein the substance comprises an antigen, and wherein each detection region includes an inlet for introducing an antibody into that detection region.
 20. The apparatus of claim 19 wherein a plurality of the detection regions are arranged for parallel detection of a plurality of different substances without cross-contamination.
 21. The apparatus of claim 19 wherein the detection regions are arranged for parallel detection of a plurality of different antigens without cross-contamination.
 22. The apparatus of claim 14 wherein the channel is angled such that a plurality of the detection regions are not aligned along a common axis.
 23. The apparatus of claim 1 wherein the first channel region comprises a plurality of focusing regions, each focusing region including its own subset of the first plurality of electrodes, wherein each subset of the first plurality of electrodes comprises an opposing pair of electrodes in a rampdown orientation to define a rampdown channel within its focusing region.
 24. The apparatus of claim 23 wherein a plurality of the focusing regions are arranged in series.
 25. The apparatus of claim 23 wherein a plurality of the focusing regions are arranged in parallel.
 26. The apparatus of claim 1 further comprising: an antibody that promotes attachment of the substance to the second plurality of electrodes.
 27. The apparatus of claim 26 wherein the antibody is resident in the second channel region.
 28. The apparatus of claim 26 wherein the antibody promotes attachment of bacteria to the second plurality of electrodes, and wherein the bacteria serves as the substance.
 29. The apparatus of claim 28 wherein the antibody promotes attachment of bacteria of the family Enterobacteriaceae to the second plurality of electrodes.
 30. The apparatus of claim 29 wherein the antibody promotes attachment of bacteria of the family Enterobacteriaceae selected from the group consisting of Escherichia, Klebsiella, Proteus, Enterobacter, Aerobacter, Serratia, Providencia, Citrobacter, Morganella, Yersinia, Envinia, Shigella, Salmonella, and combinations thereof to the second plurality of electrodes.
 31. The apparatus of claim 28 wherein the antibody promotes attachment of bacteria that comprise at least one of Bacillus, Campylobacter, Listeria, Staphylococcus, Streptococcus, and Vibrio to the second plurality of electrodes.
 32. The apparatus of claim 1 wherein the channel is arranged as a microchannel; and wherein the microchannel, the first plurality of electrodes, the second plurality of electrodes, and the third plurality of electrodes are resident on a single substrate.
 33. The apparatus of claim 32 wherein the substrate has a planar dimension that defines a floor for the microchannel; wherein the first plurality of electrodes comprise an opposing pair of electrodes in a rampdown orientation that define the rampdown channel; and wherein the opposing pair of electrodes in the rampdown orientation are vertical relative to the floor.
 34. The apparatus of claim 33 wherein the first plurality of electrodes further comprise a plurality of opposing finger pair electrodes that extend outward from the opposing pair of electrodes in the rampdown orientation.
 35. The apparatus of claim 32 wherein the substrate has a planar dimension that defines a floor for the microchannel; and wherein the third plurality of electrodes are vertical relative to the floor.
 36. The apparatus of claim 1 wherein the second plurality of electrodes are washable for reusability of the apparatus.
 37. An impedance-based sensor apparatus comprising: a channel adapted to provide a path for a flow of fluid material that comprises a substance, wherein the channel includes a first channel region and a second channel region, wherein the second channel region is downstream from the first channel region with respect to the flow path for the fluid material, and wherein first channel region is arranged as a rampdown channel; a first plurality of electrodes positioned in the first channel region, the first plurality of electrodes configured to generate a non-uniform electric field within the first channel region that produces a dielectrophoresis effect on the substance to thereby focus a flow of the fluid material into the second channel region that has a higher concentration of the substance relative to the fluid material at an inlet to the first channel region; a second plurality of electrodes positioned in the second channel region, the second plurality of electrodes configured to exhibit a change in impedance based on a concentration of the substance in the fluid material within the second channel region; and a third plurality of electrodes, the third plurality of electrodes configured to generate a non-uniform electric field within second channel region that produces a dielectrophoresis effect on the substance within the second channel region to trap a concentration of the substance in the second channel region for detection by the second plurality of electrodes; wherein the third plurality of electrodes comprise a first pair of opposing electrodes upstream from the second plurality of electrodes with respect to the flow path for the fluid material and a second pair of opposing electrodes downstream from the second plurality of electrodes with respect to the flow path for the fluid material; and wherein the first and second pairs of opposing electrodes lie in different planes that are not parallel with each other.
 38. The apparatus of claim 37 wherein the first and second pairs of opposing electrodes are orthogonal to the second plurality of electrodes. 